DETECTION OF INCORPORATED IODODEOXYURIDINE IN COLONIES BY IMMUNOPEROXIDASE STAINING - A NOVEL METHOD TO MEASURE THE PROPORTION OF CYCLING COLONY-FORMING CELLS

In vitro suicide by tritiated thymidine (H-3-TdR), Ilydroxyurea (HU), or cytosine arabinoside (Ara-C) is assumed to renect the proportion of colony-forming cells in S-phase at the time of exposure. However, the se techniques are not always accurate. Nonradioactive iododeoxyuridine (IdUrd) is incorporated into DNA during S-phase and can be detected b y monoclonal antibodies. In the present study, a new IdUrd application was developed to investigate the kinetics of hematopoietic progenitor cells. After incubation with IdUrd, colony-forming cells were culture d in semisolid assay. An immunoperoxidase staining protocol was develo ped to detect IdUrd in cells of colonies in agar. Colony-forming cells in S-phase during the IdUrd exposure were postulated to give rise to IdUrd(+) colonies, whereas non-S-phase cells would generate IdUrd(-) c olonies. Toxicity, sensitivity, and IdUrd inactivation studies indicat ed that progenitor cells could safely be pulse-labeled for 2 hours wit h 40 mu M IdUrd, whereas prolonged labeling with 1 mu M IdUrd was at l east feasible for 5 days. Molt-4 cells and normal bone marrow cells we re used to compare IdUrd pulse-labeling with H-3-TdR suicide. Part of the Molt-4 cells were enriched for G(1)- and S-phase cells by countern ow centrifugation. The bone marrow cells were either unstimulated or s timulated with growth factors. As a result, the accuracy of both techn iques could be tested in populations with different quantities of S-ph ase cells. Wide confidence intervals of the suicide technique contrast ed with the small confidence intervals obtained with IdUrd pulse-label ing. For instance, the fraction of Molt-4 cells with 27.8% S-phase cel ls contained 17.7% (confidence interval -8.2 to 43.6%) clonogenic cell s in S-phase when determined with H-3-TdR suicide. Of this fraction, t he percentage of clonogenic cells in S-phase was 30.6% with a confiden ce interval of 25.5 to 36.2% when determined with IdUrd pulse-labeling . In our hands, the IdUrd pulse-labeling was more accurate than the H- 3-TdR suicide technique. Thus far, kinetic studies of progenitors have been limited to the determination of the fraction of S-phase cells by suicide techniques. By prolonged IdUrd labeling, it is now possible t o determine the proliferating fraction of progenitor cells.